Reovirus Neuropathogenesis

呼肠孤病毒神经发病机制

基本信息

批准号：
10607594
负责人：
TERENCE S. DERMODY
金额：
$ 56.55万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-22 至 2027-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10607594
关键词：
3-Dimensional Adult Afferent Neurons Atomic Force Microscopy Axon Axonal Transport Binding Biophysics Brain Brain imaging CRISPR-mediated transcriptional activation Capsid Capsid Proteins Cells Cerebellum Child Clustered Regularly Interspaced Short Palindromic Repeats Cryoelectron Microscopy Cultured Cells Development Disease Double Stranded RNA Virus Dynein ATPase Electron Microscopy Encephalitis Engineering Fiber Genome Hippocampus (Brain)Human Immunocompetent Immunoglobulins In Vitro Infection Integration Host Factors Integrins Interneurons Intervention Intramuscular Knockout Mice Knowledge Malignant Neoplasms Mammals Mediating Morbidity - disease rate Motor Mus Mutagenesis Nervous system structure Neuraxis Neurons Neuropathogenesis Neurotropism Newborn Infant Oncolytic Pattern Peripheral Pharmacology Population Process Proteins Reovirus Reovirus Infections Research Resolution Role Route Sensory Receptors Series Serotyping Structure Synapses System Testing Thalamic structure Tissues Tropism Viral Viral Encephalitis Viral Pathogenesis Virulence Virus Work blind cell type clinical development experimental study fast axonal transport in vivo mortality mutant nervous system disorder neural circuit neuronal cell body neurotropic neurotropic virus neurovirulence new therapeutic target oncolytic vector particle receptor receptor function reconstruction recruit relating to nervous system reverse genetics screening targeted treatment transmission process uptake virus host interaction

项目摘要

Viral encephalitis is an important cause of morbidity and mortality in children and adults. Key knowledge gaps about the pathogenesis of viral encephalitis include how neurotropic viruses target the central nervous system (CNS), internalize into neurons, and disseminate in the brain. The proposed research uses reovirus, a genetically tractable double-stranded RNA virus that causes encephalitis and shows promise as an oncolytic agent, to dissect mechanisms of viral tropism, cell entry, and spread in the CNS. Reovirus strains display serotype-specific patterns of systemic dissemination and neurologic disease. Serotype 3 (T3) reoviruses are exquisitely neurotropic, internalize into neurons using macropinocytosis, and spread intracellularly by fast axonal transport. Reovirus uses β1 integrin to internalize into some types of cells, but its function in neural cell entry is unknown. Other previously identified reovirus receptors are dispensable for reovirus infection in the murine CNS. We recently identified a new reovirus receptor, paired immunoglobulin-like receptor B (PirB), that allows serotype-independent reovirus infection of cultured cells but is required for efficient T3 infection of neurons and full neurovirulence. Three integrated specific aims are proposed to enhance knowledge of reovirus neuropathogenesis that may have broader applications to other neurotropic viruses. In Specific Aim 1, functions of host receptors in reovirus neuropathogenesis will be determined. Biophysical interactions between PirB and reovirus will be assessed using cryo-electron microscopy, atomic-force microscopy, and structure- guided mutagenesis. The function of PirB in reovirus disease will be elucidated by comparing infection and virulence in wild-type (WT) and PirB-null mice. T3 reovirus-specific receptors that couple with PirB for neurotropism will be identified using CRISPR activation screening. In Specific Aim 2, mechanisms of reovirus entry into CNS neurons will be defined. The functions of PirB and β1 integrin in reovirus neural entry will be determined using mutant receptors and receptor-blind viruses. Receptor-dependent control of macropinocytosis will be elucidated using super-resolution, live-cell, and electron microscopy. Mechanisms of dynein motor recruitment for reovirus transport within axons will be dissected by investigating interactions between WT and mutant reovirus receptors and dynein subunits. In Specific Aim 3, the basis of reovirus dissemination in the CNS will be elucidated. The role of host receptors in reovirus neural transit will be defined using whole-brain imaging and three-dimensional reconstruction to trace reovirus infection and neural spread in WT and receptor- null mice. Mechanisms of reovirus transsynaptic transmission will be elucidated using in vitro and in vivo neural circuits. The effect of altering synaptic activity on reovirus neural transit will be tested using pharmacologic intervention and chemogenetically altered mice. Taken together, these studies will define mechanisms used by neurotropic reovirus to infect and disseminate in the brain and may promote further development of reovirus oncolytics that selectively target the nervous system.

病毒脑炎是儿童和成人发病率和死亡率的重要原因。关键知识差距关于病毒脑炎的发病机理包括神经性病毒的靶向中枢神经系统（CNS），内部化为神经元，并在大脑中传播。拟议的研究使用了依孢病毒，可引起脑炎的遗传诱导双链RNA病毒试剂，剖析病毒疗法的机制，细胞进入并扩散在中枢神经系统中。葡萄病毒菌株显示全身传播和神经系统疾病的血清型特异性模式。血清型3（T3）蠕虫病毒是精美的神经性，使用大型细胞增多症内化为神经元，并通过快速扩散轴突运输。葡萄病毒使用β1整合素将某些类型的细胞内化，但其功能在神经细胞中条目未知。其他先前鉴定的依孢病毒受体对于依孢病毒感染的可分配鼠CNS。我们最近确定了一种新的葡萄病毒受体，配对的免疫球蛋白样受体B（PIRB），它是允许培养细胞的非依赖性血清型依赖病毒感染，但需要有效的T3感染神经元和完整的神经病毒。提出了三个集成的特定目的，以增强葡萄病毒的知识神经病生成可能对其他神经性病毒具有更广泛的应用。在特定的目标1中，将确定宿主受体在神经病发生中的功能。两者之间的生物物理相互作用 PIRB和异病毒将使用冷冻电子显微镜，原子力显微镜和结构 - 引导诱变。通过比较感染和野生型（WT）和PIRB-NULL小鼠中的病毒。 T3异病毒特异性受体，夫妇与PIRB 将使用CRISPR激活筛选来鉴定神经性。在特定的目标2中，葡萄病毒的机制将定义进入CNS神经元的进入。 piRB和β1整合素在异病毒神经元进入中的功能将是使用突变受体和受体盲病毒确定。受体依赖性对大型细胞增多症的控制将使用超分辨率，活细胞和电子显微镜阐明。动力蛋白电机的机理轴突内的葡萄病毒运输的募集将通过研究WT和突变型葡萄病毒受体和动力蛋白亚基。在特定的目标3中，葡萄病毒传播的基础中枢神经系统将被阐明。将使用全脑定义宿主受体在eo虫病毒神经传输中的作用成像和三维重建，以追踪蠕虫病毒感染和神经传播在WT和受体中空老鼠。将使用体外和体内中性阐明异毒病毒的转染病毒的机理电路。将使用药理学测试改变突触病毒神经传输的突触病毒神经传输的影响干预和化学发生的小鼠。综上所述，这些研究将定义由神经旋转病毒病毒以感染和传播在大脑中，并可能促进依肠病毒的进一步发展有选择地针对神经系统的癌术。